U.S. patent number 8,765,380 [Application Number 13/618,334] was granted by the patent office on 2014-07-01 for bead emulsion nucleic acid amplification.
This patent grant is currently assigned to 454 Life Sciences Corporation. The grantee listed for this patent is Jan Berka, Yi-Ju Chen, John H. Leamon, Steve Lefkowitz, Kenton L. Lohman, Vinod B. Makhijani, Jonathan M. Rothberg, Gary J. Sarkis, Maithreyn Srinivasan, Michael P. Weiner. Invention is credited to Jan Berka, Yi-Ju Chen, John H. Leamon, Steve Lefkowitz, Kenton L. Lohman, Vinod B. Makhijani, Jonathan M. Rothberg, Gary J. Sarkis, Maithreyn Srinivasan, Michael P. Weiner.
United States Patent |
8,765,380 |
Berka , et al. |
July 1, 2014 |
Bead emulsion nucleic acid amplification
Abstract
Disclosed are methods for nucleic acid amplification wherein
nucleic acid templates, beads, and amplification reaction solution
are emulsified and the nucleic acid templates are amplified to
provide clonal copies of the nucleic acid templates attached to the
beads. Also disclosed are kits and apparatuses for performing the
methods of the invention.
Inventors: |
Berka; Jan (New Haven, CT),
Chen; Yi-Ju (New Haven, CT), Leamon; John H. (Guilford,
CT), Lefkowitz; Steve (Branford, CT), Lohman; Kenton
L. (Guilford, CT), Makhijani; Vinod B. (Guilford,
CT), Rothberg; Jonathan M. (Guilford, CT), Sarkis; Gary
J. (Guilford, CT), Srinivasan; Maithreyn (Branford,
CT), Weiner; Michael P. (Guilford, CT) |
Applicant: |
Name |
City |
State |
Country |
Type |
Berka; Jan
Chen; Yi-Ju
Leamon; John H.
Lefkowitz; Steve
Lohman; Kenton L.
Makhijani; Vinod B.
Rothberg; Jonathan M.
Sarkis; Gary J.
Srinivasan; Maithreyn
Weiner; Michael P. |
New Haven
New Haven
Guilford
Branford
Guilford
Guilford
Guilford
Guilford
Branford
Guilford |
CT
CT
CT
CT
CT
CT
CT
CT
CT
CT |
US
US
US
US
US
US
US
US
US
US |
|
|
Assignee: |
454 Life Sciences Corporation
(Branford, CT)
|
Family
ID: |
32854649 |
Appl.
No.: |
13/618,334 |
Filed: |
September 14, 2012 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130078638 A1 |
Mar 28, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13033240 |
Feb 23, 2011 |
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11982095 |
Oct 31, 2007 |
8012690 |
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10767899 |
Jan 28, 2004 |
7842457 |
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60443471 |
Jan 29, 2003 |
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60465071 |
Apr 23, 2003 |
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60476313 |
Jun 6, 2003 |
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60476504 |
Jun 6, 2003 |
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60476592 |
Jun 6, 2003 |
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60476602 |
Jun 6, 2003 |
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60497985 |
Aug 25, 2003 |
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Current U.S.
Class: |
435/6.12;
435/91.2 |
Current CPC
Class: |
C12Q
1/6874 (20130101); C12Q 1/6865 (20130101); C12N
15/1075 (20130101); B01L 3/502707 (20130101); C12Q
1/6867 (20130101); C12Q 1/6806 (20130101); C12Q
1/686 (20130101); C12Q 1/6834 (20130101); G01R
33/1269 (20130101); C12Q 1/6869 (20130101); G01N
21/6452 (20130101); C12N 15/1093 (20130101); G01N
21/6428 (20130101); B01L 3/502715 (20130101); C12Q
1/6844 (20130101); C07H 21/00 (20130101); G01N
21/253 (20130101); C12Q 1/6834 (20130101); C12Q
2565/119 (20130101); C12Q 2531/113 (20130101); C12Q
1/686 (20130101); C12Q 2565/119 (20130101); C12Q
1/686 (20130101); C12Q 2565/537 (20130101); C12Q
2547/107 (20130101); C12Q 2547/101 (20130101); C12Q
1/686 (20130101); C12Q 2537/143 (20130101); C12Q
2527/125 (20130101); C12Q 1/6869 (20130101); C12Q
2525/186 (20130101); C12Q 2565/301 (20130101); C12Q
2565/537 (20130101); C12Q 1/6869 (20130101); C12Q
2565/537 (20130101); C12Q 2565/301 (20130101); C12Q
2563/149 (20130101); C12Q 1/6874 (20130101); C12Q
2565/301 (20130101); C12Q 2531/113 (20130101); C12Q
1/6844 (20130101); C12Q 2531/125 (20130101); C12Q
2565/119 (20130101); C12Q 2527/125 (20130101); C12Q
1/686 (20130101); C12Q 2547/101 (20130101); C12Q
2565/119 (20130101); C12Q 2527/125 (20130101); C12Q
1/6865 (20130101); C12Q 2547/101 (20130101); C12Q
2565/119 (20130101); C12Q 2527/125 (20130101); C12Q
1/6867 (20130101); C12Q 2547/101 (20130101); C12Q
2565/119 (20130101); C12Q 2527/125 (20130101); C12Q
1/6869 (20130101); C12Q 2537/149 (20130101); C12Q
2535/119 (20130101); C12Q 2525/186 (20130101); B01L
7/52 (20130101); G01N 21/6458 (20130101); Y02P
20/582 (20151101); G01N 2021/6484 (20130101); Y10T
436/143333 (20150115); B01L 2300/0636 (20130101); B01L
3/5027 (20130101); B01L 2300/0819 (20130101); B01L
3/5085 (20130101); B01L 2300/0877 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12P 19/34 (20060101) |
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|
Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Thomas; David
Attorney, Agent or Firm: Cooley LLP
Parent Case Text
RELATED APPLICATIONS
This application claims the benefit of priority to the following
applications: U.S. Ser. No. 60/443,471 filed Jan. 29, 2003, U.S.
Ser. No. 60/465,071 filed Apr. 23, 2003; U.S. Ser. No. 60/476,313
filed on Jun. 6, 2003, U.S. Ser. No. 60/476,504 filed Jun. 6, 2003,
U.S. Ser. No. 60/476,592 filed on Jun. 6, 2003; U.S. Ser. No.
60/476,602 filed on Jun. 6, 2003, and U.S. Ser. No. 60/497,985
filed Aug. 25, 2003. All patent and patent applications in this
paragraph are hereby fully incorporated herein by reference.
This application also incorporates by reference the following U.S.
patent applications: "Method For Preparing Single-Stranded DNA
Libraries" filed Jan. 28, 2004 as U.S. Ser. No. 10/767,894, now
abandoned, "Double Ended Sequencing" filed Jan. 28, 2004 as U.S.
Ser. No. 10/768,729, which issued as U.S. Pat. No. 7,244,567 on
Jul. 17, 2007, and "Methods Of Amplifying And Sequencing Nucleic
Acids" filed Jan. 28, 2004 as U.S. Ser. No. 10/767,379, which
issued as U.S. Pat. No. 7,323,305 on Jan. 29, 2008.
Claims
What is claimed is:
1. A method for analyzing nucleic acid sequences comprising: (a)
delivering a plurality of molecules of a deoxyribonucleic acid into
aqueous microreactors in a water-in-oil emulsion such that a
plurality of aqueous microreactors comprise a single molecule of
the deoxyribonucleic acid, a single bead capable of hybridizing the
deoxyribonucleic acid, and reagents necessary to perform
deoxyribonucleic acid amplification; (b) amplifying the
deoxyribonucleic acid in the microreactors to form amplified copies
of said deoxyribonucleic acid bound to beads in the microreactors;
(c) determining the presence of amplified copies of said
deoxyribonucleic acid bound to a bead.
2. The method of claim 1 wherein step (c) is accomplished using
polymerase chain reaction.
3. The method of claim 1, wherein a majority of the microcarriers
include a single molecule of the deoxyribonucleic acid.
4. The method of claim 1, wherein said reagents include a
polymerase chain reaction solution further comprising nucleotide
triphosphates, a thermostable polymerase, and a buffer compatible
with polymerase chain reaction conditions.
5. The method of claim 1, wherein said emulsion is heat stable.
6. The method of claim 1, wherein amplification is carried out by a
method selected from the group consisting of transcription-based
amplification, rapid amplification of cDNA ends, continuous flow
amplification, and rolling circle amplification.
7. The method of claim 1, performed with at least 50,000 molecules
of deoxyribonucleic acid.
8. The method of claim 1, wherein between at least 1 to 20,000,000
copies of each deoxyribonucleic acid molecule are bound to each
bead.
9. The method of claim 1, further comprising, after step (b), the
step of: separating the beads with amplified copies of the
deoxyribonucleic acid thereon away from the beads that do not have
amplified copies of the deoxyribonucleic acid bound.
Description
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
The contents of the text file named "21465-508C03US_ST25.txt",
which was created on Nov. 28, 2012 and is 3 KB in size, are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates to methods for amplifying nucleic
acid templates from low copy number to quantities amenable for
sequencing on a solid support such as a bead. The present invention
is also directed to zero bead removal--a method of enriching for
solid support containing amplified nucleic acids is also
disclosed.
BACKGROUND
The ability to amplify a plurality of nucleic acid sequences, such
as a genomic library or a cDNA library, is critical given the
inefficiency of current methods of sequencing. Current sequencing
technologies require millions of copies of nucleic acid per
sequencing reaction. Furthermore, the sequencing of a human genome
would require about tens of millions of different sequencing
reactions. If the starting material is limited, amplification of
the initial DNA is necessary before genomic sequencing. The
starting material may be limited, for example, if the genome to be
sequenced is from a trace quantity of pathogen or from a prenatal
patient. Current techniques for in vitro genome amplification
involve laborious cloning and culturing protocols that have limited
the utility of genomic sequencing. Other techniques, such as PCR,
while fast and reliable, are unable to amplify a genome in a
representative fashion.
While random primed PCR can be easily engineered to amplify a
plurality of nucleic acids in one reaction, this method is not
preferred because the amplified library is not representative of
the starting library. That is, in a random PCR environment, some
DNA sequences are preferentially amplified at the expense of other
sequences such that the amplified product does not represent the
starting material. This problem with PCR may be overcome if each
individual member of a library is amplified in a separate reaction.
However, this approach may be impractical if many thousands of
separate reaction tubes are required for the amplification process,
as a genomic library or cDNA library may include more than 100,000
fragments. Individual amplification of each fragment of these
libraries in separate reaction is not practical.
SUMMARY OF THE INVENTION
The present invention provides for a method of amplifying a
plurality of nucleic acids (e.g., each sequence of a DNA library,
transcriptome, or genome) in a rapid and economical manner in a
single reaction tube. One use of the method of the invention is to
perform simultaneous clonal amplification (e.g., by PCR) of a
plurality of samples (as many as several hundred thousand) in one
reaction vessel. This invention further provides means for
encapsulating a plurality of DNA samples individually in a
microcapsule of an emulsion (i.e., a microreactor), performing
amplification of the plurality of encapsulated nucleic acid samples
simultaneously, and releasing said amplified plurality of DNA from
the microcapsules for subsequent reactions.
In one embodiment, single copies of the nucleic acid template
species are hybridized to capture beads comprising, e.g., capture
oligonucleotides or chemical groups that bind to the nucleic acid
template. The beads are suspended in complete amplification
solution (see Example 2 for an example of an amplification
solution) and emulsified to produce microreactors (typically 100 to
200 microns in diameter). After this, amplification (e.g., PCR) is
used to clonally increase copy number of the initial template
species in the microreactors, and these copies bind to the capture
beads in the microreactors.
In an alternate embodiment, capture beads are added to an
amplification reaction mixture (e.g., an amplification solution
from Example 2) comprising nucleic acid template and this mixture
is emulsified to produce microreactors. Amplification (e.g., PCR)
is used to clonally increase copy number of the initial template
species in the microreactors, and these copies bind to the capture
beads in the microreactors.
One advantage of the present invention is that the microreactors
allow the simultaneous clonal and discrete amplification of many
different templates without cross contamination of the amplified
products or reagents, or domination of one particular template or
set of templates (e.g., PCR bias). The amplification reaction, for
example, may be performed simultaneously with at least 3,000
microreactors per microliter of reaction mix. Preferably, each
microreactor comprises one or fewer species of amplified
template.
In various embodiments of the invention, the microreactors have an
average size of about 10 .mu.m to about 250 .mu.m. In a preferred
embodiment, the microreactors have an average diameter of about 60
to about 200 .mu.m. In a more preferred embodiment, the
microreactors have an average diameter of about 60 .mu.m, such as
an average of 40 .mu.m to 80 .mu.m in diameter. In an embodiment,
the microreactors have an average diameter of about 60 .mu.m. In
another preferred embodiment, the microreactors have an average
volume of about 113 pl. In a most preferred embodiment, about 3000
microreactors are contained within a microliter of a 1:2 water to
oil emulsion.
The present invention also provides for a method for producing a
plurality of nucleic acid template-carrying beads wherein each bead
comprises up to and more than 1,000,000 copies of a single nucleic
acid sequence. In one preferred embodiment, each bead may comprise
over 20 million copies of a single nucleic acid.
The present invention further provides for a library made by the
methods of the invention. The library may be made by using, e.g., a
genomic DNA library, a cDNA library, or a plasmid library as the
starting material for amplification. The library may be derived
from any population of nucleic acids, e.g., biological or synthetic
in origin.
The present invention also provides for a method of enriching for
those beads that contains the product of successful DNA
amplification (i.e., by removing beads that have no DNA attached
thereto).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Schematic of the structure of a DNA capture bead.
FIGS. 2A-2B Schematic of one embodiment of a bead emulsion
amplification process.
FIG. 3 Schematic of an enrichment process to remove beads that do
not have any DNA attached thereto.
FIG. 4 Depiction of jig used to hold tubes on the stir plate below
vertical syringe pump. The jig was modified to hold three sets of
bead emulsion amplification reaction mixtures. The syringe was
loaded with the PCR reaction mixture and beads.
FIG. 5 Depiction of optimal placement of syringes in vertical
syringe pump and orientation of emulsion tubes below syringe
outlets.
FIG. 6 Depiction of optimal placement of syringe pump pusher block
against syringe plungers, and optimal orientation of jig on the
stir plate. Using this arrangement, the syringe contents were
expelled into the agitated emulsion oil.
FIG. 7 Depiction of beads (see arrows) suspended in individual
microreactors according to the methods of the invention.
FIGS. 8A-8C Schematic showing the initial stages of bead emulsion
amplification used in conjunction with double ended sequencing. The
NHS-activated bead (FIG. 8A) is attached with capture primers (FIG.
8B), and encapsulated in a microreactor comprising the DNA capture
bead and template (FIG. 8C).
FIG. 9 Schematic showing the amplification and capture stages of
bead emulsion amplification used in conjunction with double ended
sequencing. The template is amplified by solution phase PCR and the
amplification products are attached to the DNA capture bead.
FIG. 10 Schematic showing the later stages of bead emulsion
amplification used in conjunction with double ended sequencing. The
emulsion is broken down (FIGS. 10A-10B), the second strand of the
amplification product is removed and enrichment is used to maximize
the number of beads bound with amplification product (FIG. 10C),
the sequencing primers are annealed (FIG. 10D), and the first
strand is sequenced (FIG. 10E), followed by the second strand.
DETAILED DESCRIPTION OF INVENTION
Brief Overview of Bread Emulsion Amplification
A brief overview of one embodiment of the invention is discussed
below. A more detailed description of each individual step of this
embodiment will follow. In this embodiment, PCR is the chosen
amplification technique.
In one aspect of the invention, bead emulsion amplification is
performed by attaching a template (e.g., DNA template) to be
amplified to a solid support, preferably in the form of a generally
spherical bead. The bead is linked to a large number of a single
primer species (i.e., primer B in FIG. 2) that is complementary to
a region of the template DNA and the amplification copies of this
template. Alternately, the bead is linked, to chemical groups
(e.g., biotin) that can bind to chemical groups (e.g.,
streptavidin) included on the template DNA and amplification copies
of this template. The beads are suspended in aqueous reaction
mixture and then encapsulated in a water-in-oil emulsion. In
different aspects of the invention, the template DNA is bound to
the bead prior to emulsification, or the template DNA is included
in solution in the amplification reaction mixture. In a preferred
embodiment, an amplification step is performed prior to
distribution of the nucleic acid templates onto a multiwell (e.g.,
picotiter) plate.
In certain embodiments, the emulsion is composed of discrete
aqueous phase microdroplets, e.g., averaging approximately 60 to
200 .mu.m in diameter, enclosed by a thermostable oil phase. Each
microdroplet contains, preferably, amplification reaction solution
(i.e., the reagents necessary for nucleic acid amplification). An
example of an amplification reaction solution would be a PCR
reaction mixture (polymerase, salts, dNTPs; see Example 2 for an
example of one embodiment) and a pair of PCR primers (primer A and
primer B). See, FIG. 2A. In some cases, the template DNA is
included in the reaction mixture. A subset of the microdroplet
population includes the DNA bead and the template. This subset of
microdroplet is the basis for the amplification. The remaining
microcapsules do not contain template DNA and will not participate
in amplification. In one embodiment, the amplification technique is
PCR and the PCR primers are present in an 8:1 or 16:1 ratio (i.e.,
8 or 16 of one primer to 1 of the second) primer) to perform
asymmetric PCR. In another embodiment, the ratio of PCR primers may
be substantially equal for normal PCR.
The amplification reaction, such as PCR, may be performed using any
suitable method. In the following overview, one mechanism of PCR is
discussed as an illustration. However, it is understood that the
invention is not limited to this mechanism. In the example, a
region of the DNA molecule (B' region) is annealed to an
oligonucleotide immobilized to a bead (primer B). During
thermocycling (FIG. 2B), the bond between the single stranded
template and the immobilized B primer on the bead is broken,
releasing the template into the surrounding microencapsulated
solution. The amplification solution, in this case, the PCR
solution, contains addition solution phase primer A and primer B
(e.g., in a 8:1 or 16:1 ratio). Solution phase B primers readily
bind to the complementary B' region of the template as binding
kinetics are more rapid for solution phase primers than for
immobilized primers.
In early phase PCR, both A and B strands amplify equally well (FIG.
2C). By midphase PCR (i.e., between cycles 10 and 30) the B primers
are depleted, halting exponential amplification. The reaction then
enters asymmetric amplification and the amplicon population becomes
dominated by A strands (FIG. 2D). In late phase PCR (FIG. 2E),
after 30 to 40 cycles, asymmetric amplification increases the
concentration of A strands in solution. Excess A strands begin, to
anneal to bead immobilized B primers. Thermostable polymerases then
utilize the A strand as a template to synthesize an immobilized,
bead bound B strand of the amplicon.
In final phase PCR (FIG. 2F), continued thermal cycling forces
additional annealing to bead bound primers. Solution phase
amplification may be minimal at this stage but concentration of
immobilized B strands increase. Then, the emulsion is broken and
the immobilized product is rendered single stranded by denaturing
(by heat, pH etc.) which removes the complimentary A strand. The A
primers are annealed to the A' region of immobilized strand, and
immobilized strand is loaded with sequencing enzymes, and any
necessary accessory proteins. The beads are then sequenced using
recognized pyrophosphate techniques (described, e.g., in U.S. Pat.
Nos. 6,274,320, 6,258,568 and 6,210,891, incorporated in toto
herein by reference).
Template Design
In a preferred embodiment, the nucleic acid template to be
amplified by bead emulsion amplification is a population of DNA
such as, for example, a genomic DNA library or a cDNA library. It
is preferred that each member of the DNA population have a common
nucleic acid sequence at the first end and a common nucleic acid
sequence at a second end. This can be accomplished, for example, by
ligating a first adaptor DNA sequence to one end and a second
adaptor DNA sequence to a second end of each member of the DNA
population. Many DNA and cDNA libraries, by nature of the cloning
vector (e.g., Bluescript, Stratagene, La Jolla, Calif.) fit this
description of having a common sequence at a first end and a second
common sequence at a second end of each member DNA. The nucleic
acid template may be of any size amenable to in vitro amplification
(including the preferred amplification techniques of PCR and
asymmetric PCR). In a preferred embodiment, the template is about
150 to 750 bp in size, such as, for example about 250 bp in
size.
Binding Nucleic Acid Template to Capture Beads
In one aspect of the invention, a single stranded nucleic acid
template to be amplified is attached to a capture bead. The
template may be captured to the bead prior to emulsification or
after the emulsion has been formed. In a preferred aspect, the
amplification copies of the nucleic acid template are attached to a
capture bead. As non-limiting examples, these attachments may be
mediated by chemical groups or oligonucleotides that are bound to
the surface of the bead. The nucleic acid (e.g., the nucleic acid
template, amplification copies, or oligonucleotides) may be
attached to the solid support (e.g., a capture bead) in any manner
known in the art.
According to the present invention, covalent chemical attachment of
a nucleic acid to the bead can be accomplished by using standard
coupling agents. For example, water-soluble carbodiimide can be
used to link the 5'-phosphate of a DNA sequence to amine-coated
capture beads through a phosphoamidate bond. Alternatively,
specific oligonucleotides can be coupled to the bead using similar
chemistry, and to then DNA ligase can be used to ligate the DNA
template to the oligonucleotide on the bead. Other linkage
chemistries to join the oligonucleotide to the beads include the
use of N-hydroxysuccinamide (NHS) and its derivatives.
In an exemplary method, one end of a linker may contain a reactive
group (such as an amide group) which forms a covalent bond with the
solid support, while the other end of the linker contains a second
reactive group that can bond with the oligonucleotide to be
immobilized. In a preferred embodiment, the oligonucleotide is
bound to the DNA capture bead by covalent linkage. However,
non-covalent linkages, such as chelation or antigen-antibody
complexes, may also be used to join the oligonucleotide to the
bead.
As non-limiting examples, oligonucleotides can be employed which
specifically hybridize to unique sequences at the end of the DNA
fragment, such as the overlapping end from a restriction enzyme
site or the "sticky ends" of cloning vectors, but blunt-end linkers
can also be used. These methods are described in detail in U.S.
Pat. No. 5,674,743. It is preferred that the beads will continue to
bind the immobilized oligonucleotide throughout the steps in the
methods of the invention.
In one embodiment of the invention, each capture bead is designed
to have a plurality of oligonucleotides that recognize (i.e., are
complementary to) a portion of the nucleic template, and the
amplification copies of this template. In the methods described
herein, clonal amplification of the template species is desired, so
it is preferred that only one unique nucleic acid species is
attached to any one capture bead.
The beads used herein may be of any convenient size and fabricated
from any number of known materials. Example of such materials
include: inorganics, natural polymers, and synthetic polymers.
Specific examples of these materials include: cellulose, cellulose
derivatives, acrylic resins, glass, silica gels, polystyrene,
gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and
acrylamide, polystyrene cross-linked with divinylbenzene or the
like (as described, e.g., in Merrifield, Biochemistry 1964, 3,
1385-1390), polyacrylamides, latex gels, polystyrene, dextran,
rubber, silicon, plastics, nitrocellulose, natural sponges, silica
gels, control pore glass, metals, cross-linked dextrans (e.g.,
Sephadex.TM.) agarose gel (Sepharose.TM.), and other solid phase
supports known to those of skill in the art. In preferred
embodiments, the capture beads are beads approximately 2 to 100
.mu.m in diameter, or 10 to 80 .mu.m in diameter, most preferably
20 to 40 .mu.m in diameter. In a preferred embodiment, the capture
beads are Sepharose beads.
Emulsification
For use with the present invention, capture beads with or without
attached nucleic acid template are suspended in a heat stable
water-in-oil emulsion. It is contemplated that a plurality of the
microreactors include only one template and one bead. There may be
many droplets that do not contain a template or which do not
contain a bead. Likewise there may be droplets that contain more
than one copy of a template. The emulsion may be formed according
to any suitable method known in the art. One method of creating
emulsion is described below but any method for making an emulsion
may be used. These methods are known in the art and include
adjuvant methods, counter-flow methods, cross-current methods,
rotating drum methods, and membrane methods. Furthermore, the size
of the microcapsules may be adjusted by varying the flow rate and
speed of the components. For example, in dropwise addition, the
size of the drops and the total time of delivery may be varied.
Preferably, the emulsion contains a density of about 3,000 beads
encapsulated per microliter.
Various emulsions that are suitable for biologic reactions are
referred to in Griffiths and Tawfik, EMBO, 22, pp. 24-35 (2003);
Ghadessy et al., Proc. Natl. Acad. Sci. USA 98, pp. 4552-4557
(2001); U.S. Pat. No. 6,489,103 and WO 02/22869, each fully
incorporated herein by reference. It is noted that Griffiths et
al., (U.S. Pat. No. 6,489,103 and WO 99/02671) refers to a method
for in vitro sorting of one or more genetic elements encoding a
gene products having a desired activity. This method involves
compartmentalizing a gene, expressing the gene, and sorting the
compartmentalized gene based on the expressed product. In contrast
to the present invention, the microencapsulated sorting method of
Griffith is not suitable for parallel analysis of multiple
microcapsules because their nucleic acid product is not anchored
and cannot be anchored. Since the nucleic acids of Griffiths are
not anchored, they would be mixed together during
demulsification.
The emulsion is preferably generated by adding beads to an
amplification solution. As used herein, the term "amplification
solution" means the sufficient mixture of reagents that is
necessary to perform amplification of template DNA. One example of
an amplification solution, a PCR amplification solution, is
provided in the Examples below. It will be appreciated that various
modifications may be made to the amplification solution based on
the type of amplification being performed and whether the template
DNA is attached to the beads or provided in solution. In one
embodiment, the mixture of beads and amplification solution is
added dropwise into a spinning mixture of biocompatible oil (e.g.,
light mineral oil, Sigma) and allowed to emulsify. In another
embodiment, the beads and amplification solution are added dropwise
into a cross-flow of biocompatible oil. The oil used may be
supplemented with one or more biocompatible emulsion stabilizers.
These emulsion stabilizers may include Atlox 4912, Span 80, and
other recognized and commercially available suitable stabilizers.
In preferred aspects, the emulsion is heat stable to allow thermal
cycling, e.g., to at least 94.degree. C., at least 95.degree. C.,
or at least 96.degree. C. Preferably, the droplets formed range in
size from about 5 microns to about 500 microns, more preferably
from about 10 microns to about 350 microns, even more preferably
from about 50 to 250 microns, and most preferably from about 100
microns to about 200 microns. Advantageously, cross-flow fluid
mixing allows for control of the droplet formation, and uniformity
of droplet size. We note that smaller water droplets not containing
beads may be present in the emulsion.
The microreactors should be sufficiently large to encompass
sufficient amplification reagents for the degree of amplification
required. However, the microreactors should be sufficiently small
so that a population of microreactors, each containing a member of
a DNA library, can be amplified by conventional laboratory
equipment, e.g., PCR thermocycling equipment, test tubes,
incubators and the like. Notably, the use of microreactors allows
amplification of complex mixtures of templates (e.g., genomic DNA
samples or whole cell RNA) without intermixing of sequences, or
domination by one or more templates (e.g., PCR selection bias; see,
Wagner et al., 1994, Suzuki and Giovannoni, 1996; Chandler et al.,
1997, Polz and Cavanaugh, 1998).
With the limitations described above, the optimal size of a
microreactor may be on average 100 to 200 microns in diameter.
Microreactors of this size would allow amplification of a DNA
library comprising about 600,000 members in a suspension of
microreactors of less than 10 ml in volume. For example, if PCR is
the chosen amplification method, 10 ml of microreactors would fit
into 96 tubes of a regular thermocycler with 96 tube capacity. In a
preferred embodiment, the suspension of 600,000 microreactors would
have a volume of less than 1 ml. A suspension of less than 1 ml may
be amplified in about 10 tubes of a conventional PCR thermocycler.
In a most preferred embodiment, the suspension of 600,000
microreactors would have a volume of less than 0.5 ml.
Another embodiment of the invention is directed to a method of
performing nucleic acid amplification with a template and a bead,
but without attachment of the template to the bead. In one aspect,
the bead may comprise a linker molecule that can bind the amplified
nucleic acid after amplification. For example, the linker may be a
linker that can be activated. Such linkers are well known and
include temperature sensitive or salt sensitive binding pairs such
as streptavidin/biotin and antibodies/antigen. The template nucleic
acid may be encapsulated with a bead and amplified. Following
amplification, the amplified nucleic acid may be linked to the
beads, e.g., by adjustments in temperature or salt
concentration.
Amplification
After encapsulation, the template nucleic acid may be amplified,
while attached or unattached to beads, by any suitable method of
amplification including transcription-based amplification systems
(Kwoh D. et al., Proc. Natl. Acad Sci. (U.S.A.) 86:1173 (1989);
Gingeras T. R. et al., WO 88/10315; Davey, C. et al., EP
Publication No. 329,822; Miller, H. I. et al., WO 89/06700), "RACE"
(Frohman, M. A., In: PCR Protocols: A Guide to Methods and
Applications, Academic Press, NY (1990)) and one-sided PCR (Ohara,
O. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86.5673-5677 (1989)).
Still other methods such as di-oligonucleotide amplification,
isothermal amplification (Walker, G. T. et al., Proc. Natl. Acad.
Sci. (U.S.A.) 89:392-396 (1992)), Nucleic Acid Sequence Based
Amplification (NASBA; see, e.g., Deiman B et al., 2002, Mol
Biotechnol. 20(2):163-79), whole-genome amplification (see, e.g.,
Hawkins T L et al., 2002, Curr Opin Biotechnol. 13(1):65-7),
strand-displacement amplification (see, e.g., Andras S C, 2001, Mol
Biotechnol. 19(1):29-44), rolling circle amplification (reviewed in
U.S. Pat. No. 5,714,320), and other well known techniques may be
used in accordance with the present invention.
In a preferred embodiment, DNA amplification is performed by PCR.
PCR according to the present invention may be performed by
encapsulating the target nucleic acid with a PCR solution
comprising all the necessary reagents for PCR. Then, PCR may be
accomplished by exposing the emulsion to any suitable thermocycling
regimen known in the art. In a preferred embodiment, 30 to 50
cycles, preferably about 40 cycles, of amplification are performed.
It is desirable, but not necessary, that following the
amplification procedure there be one or more hybridization and
extension cycles following the cycles of amplification. In a
preferred embodiment, 10 to 30 cycles, preferably about 25 cycles,
of hybridization and extension are performed (e.g., as described in
the examples). Routinely, the template DNA is amplified until
typically at least 10,000 to 50,000,000 copies are immobilized on
each bead. It is recognized that for nucleic acid detection
applications, fewer copies of template are required. For nucleic
acid sequencing applications we prefer that at least two million to
fifty million copies, preferably about ten million to thirty
million copies of the template DNA are immobilized on each bead.
The skilled artisan will recognize that the size of bead (and
capture site thereon) determines how many captive primers can be
bound (and thus how many amplified templates may be captured onto
each bead).
PCR Primer Design
The selection of nucleic acid primers for amplification, such as
PCR amplification, is well within the abilities of one of skill in
the art, Strategies for primer design may be found throughout the
scientific literature, for example, in Rubin, E. and A. A. Levy,
Nucleic Acids Res, 1996. 24(18): p. 3538-45; and Buck, G A., et
al., Biotechniques, 1999. 27(3): p. 528-36. In a preferred
embodiment, primers can be limited to a length of 20 bases (5
tetramers) for efficient synthesis of bipartite PCRI sequencing
primers. Each primer can include a two-base GC clamp on the 5' end,
a single GC clamp on the 3' end, and all primers can share similar
T.sub.m (+/-2.degree. C.). In a preferred embodiment, hairpin
structures within the primers (internal hairpin stems
.DELTA.G>-1.9 kcal/mol) are strongly discouraged in any of the
designed primers. In another preferred embodiment, primer
dimerization is also controlled; such that a 3-base maximum
acceptable dimer is allowed. However, this is allowed to occur only
in the final six 3' bases, and the maximum allowable .DELTA.G for a
3' dimer is -2.0 kcal/mol. Preferably, a penalty is applied to
primers in which the 3' ends are too similar to others in the
group. This prevents cross-hybridization between one primer and the
reverse complement of another primer.
If the primers are designed according to the criteria described
above, the possibility of complimentary regions occurring within
the genome of interest is not of major concern, despite the
reported tolerance of PCR to mismatches in complex sample
populations (Rubin, E. and A. A. Levy. Nucleic Acids Res, 1996.
24(18): p, 3538-45). Although the probability of finding a perfect
match to a 20 base primer is extremely low (4.sup.20) (see Table
1), the probability of finding shorter non-consecutive matches
increases significantly with the size of the genome of interest. As
a result, the probability of finding a perfect match for a sequence
of at least 10 of 20 bases is 99.35% for an Adenovirus genome. The
probability of finding a perfect match for a sequence of 16 bases
is 97% for the sequences in the NCBI database (approximately 100
times more sequence information than the Adenovirus genome). The
probability of finding a perfect match for a sequence of 17 to 20
bases is 99% for the human genome (approximately 3 billion
bases).
TABLE-US-00001 TABLE 1 The probability of perfect sequence matches
for primers increases with decreasing match length requirements and
increasing size of the genome of interest. % % chance Perfect
chance for match match for in NCBI % chance probability match in
bacterial for match Match (1/ Adeno~ database~ in Human~ Length
(4{circumflex over ( )}length)) 35K bases 488M bases 3 B bases 20
9.1E-13 0.00% 0.04% 0.27% 19 7.3E-12 0.00% 0.65% 4.32% 18 4.4E-11
0.00% 5.76% 34.37% 17 2.3E-10 0.00% 35.69% 99.17% 16 1.2E-09 0.02%
97.52% >100% 15 5.6E-09 0.12% >100% >100% 14 2.6E-08 0.64%
>100% >100% 13 1.2E-07 3.29% >100% >100% 12 5.4E-57
15.68% >100% >100% 11 2.4E-06 58.16% >100% >100% 10
1.0E-05 99.35% >100% >100% 9 4.6E-05 99.77% >100% >100%
8 2.0E-04 >100% >100% >100% 7 8.5E-04 >100% >100%
>100% 6 3.7E-03 >100% >100% >100% 5 1.6E-02 >100%
>100% >100% 4 6.4E-02 >100% >100% >100% 3 2.5E-01
>100% >100% >100% 2 7.1E-01 >100% >100% >100% 1
1.0E+00 >100% >100% >100%
However, primer cross-hybridization to various regions of the
genome is less problematic than one might expect due to the random
DNA digestion used to form the nucleic acid templates. The
cross-hybridizing regions (CFHRs) are fairly benign. First, it is
unlikely that a CHR would be able to successfully compete with the
perfect match between the PCR primers in solution and the template.
In addition, any primers that include mismatches at their 3' end
will be at a significant competitive disadvantage. Even if a CHR
should out compete the intended. PCR primer, it would produce a
truncated PCR product, without a downstream site for the sequencing
primer. If the truncated product could be driven to the capture
bead and immobilized, one of two situations would result. If the
CHR out-competed the solution phase primer, then the immobilized
product would lack a sequencing primer binding site, and would
result in an empty picotiter plate (PTP) well. If the CHR
out-competed the bead-bound primer, the sequencing primer would
still be present, and the only effect would be a shorter insert.
Neither result would unduly compromise the sequencing quality.
Given the large amount of genomic material used in the sample
preparation process (currently 25 .mu.g, containing
5.29.times.10.sup.16 copies of the 35 Kb Adenovirus genome),
oversampling can be used to provide fragments that lack the
complete CHR, and allow standard PCR amplification of the region in
question.
Breaking the Emulsion and Bead Recovery
Following amplification of the nucleic acid template and the
attachment of amplification copies to the bead, the emulsion is
"broken" (also referred to as "demulsification" in the art). There
are many methods of breaking an emulsion (see, e.g., U.S. Pat. No.
5,989,892 and references cited therein) and one of skill in the art
would be able to select an appropriate method in the present
invention, one preferred method of breaking the emulsion uses
additional oil to cause the emulsion to separate into two phases.
The oil phase is then removed, and a suitable organic solvent
(e.g., hexanes added. After mixing, the oil/organic solvent phase
is removed. This step may be repeated several times. Finally, the
aqueous layers above the beads are removed. The beads are then
washed with a mixture of an organic solvent and annealing buffer
(e.g., one suitable annealing buffer is described in the examples),
and then washed again in annealing buffer. Suitable organic
solvents include alcohols such as methanol, ethanol, and the
like.
The beads bound to amplification products may then be resuspended
in aqueous solution for use, for example, in a sequencing reaction
according to known technologies. (See, Sanger, F. et al., Proc.
Natl. Acad. Sci, U.S.A. 75, 5463-5467 (1977); Maxam, A. M. &
Gilbert, W. Proc Nati Acad Sci USA 74, 560-564 (1977); Ronaghi, M.
et al., Science 281, 363, 365 (1998); Lysov, I. et al., Dokl Akad
Nauk SSSR 303, 1508-1511 (1988); Bains W. & Smith G. C. J.
Theor Biol 135, 303-307(1988); Drnanac, R. et al., Genomics 4,
114-128 (1989); Khrapko, K. R. et al., FEBS Lett 256. 118-122
(1989); Pevzner P. A. J Biomol Struct Dyn 7, 63-73 (1989);
Southern, E. M. et al., Genomics 13, 1008-1017 (1992).)
If the beads are to be used in a pyrophosphate-based sequencing
reaction (described, e.g., in U.S. Pat. Nos. 6,274,320, 6,258,568
and 6,210,891, and incorporated in toto herein by reference), then
it is necessary to remove the second strand of the PCR product and
anneal a sequencing primer to the single stranded template that is
bound to the bead. The second strand may be melted away using any
number of commonly known methods such as addition of NaOH,
application of low ionic (e.g., salt) strength, enzymatic
degradation or displacement of the second strand, or heat
processing. Following this strand removal step, the beads are
pelleted and the supernatant is discarded. The beads are
resuspended in an annealing buffer, and a sequencing primer or
other non-amplification primer is added. The primer is annealed to
the single stranded amplification product. This can be accomplished
by using an appropriate annealing buffer and temperature
conditions, e.g., as according to standard procedures in the
art.
Purifying the Beads
At this point, the amplified nucleic acid on the bead may be
sequenced either directly on the bead or in a different reaction
vessel. In an embodiment of the present invention, the nucleic acid
is sequenced directly on the bead by transferring the bead to a
reaction vessel and subjecting the nucleic acid to a sequencing
reaction (e.g., pyrophosphate or Sanger sequencing). Alternatively,
the beads may be isolated and the nucleic acid may be removed from
each bead and sequenced. In either case, the sequencing steps may
be performed on each individual bead. However, this method, while
commercially viable and technically feasible, may not be most
effective because many of the beads will be "negative" beads (i.e.,
beads without amplified nucleic acid attached). In such cases, the
optional process outlined below may be used to remove negative
beads prior to distribution onto multiwell (e.g., picotiter)
plates.
A high percentage of the beads may be negative if the goal is to
minimize the number of beads that are associated with two or more
different species of nucleic acid templates. For optimal
pyrophosphate sequencing, each bead should contain multiple copies
of a single species of nucleic acid. This can be achieved by
maximizing the total number of beads combined with a single
fragment of nucleic acid bethre amplification. For example, the
following mathematical model can be used.
For the general case of N number of DNAs randomly distributed with
M number of beads, the relative bead population associated with any
number of DNAs depends on the ratio of N/M. The fraction of beads
associated with N DNAs R(N) may be calculated using the Poisson
distribution: R(N)=exp(N/M).times.(N/M).sup.N/N! (where .times. is
the multiplication symbol)
Table 2, below, shows some calculated values for various WM (the
average DNA fragment-to-bead ratio) and N (the number of fragments
associated with a bead).
TABLE-US-00002 TABLE 2 N/M 0.1 0.5 1 2 R(0) 0.9 0.61 0.37 0.13 R(1)
0.09 0.3 0.37 0.27 R(N > 1) 0.005 0.09 0.26 0.59
In Table 2, the top row denotes the various ratios of WM. R(0)
denotes the fraction of beads with no DNA, R(1) denotes the
fraction of beads with one DNA (before amplification), and
R(N>1) denotes the fraction of DNA with more than one DNA
(before amplification).
Table 2 indicates that the maximum fraction of beads associated
with a single DNA fragment is 0.37 (37%) and this occurs at a
fragment-to-bead ratio of one-to-one. In this mixture, about 63% of
the beads cannot be used for sequencing because they are associated
with no DNA or they are associated with more than one species of
DNA. However, controlling the fragment-to-bead ratio requires
complex calculations, and variability can produce bead batches with
a significantly smaller fraction of useable beads.
This inefficiency can be significantly ameliorated if beads
containing amplicon (originating from the association with at least
one fragment) are separated from those without amplicon
(originating from beads with no associated fragments). An amplicon
is defined as any nucleic acid molecules produced by an in vitro
nucleic amplification technique. To increase efficiency, binding
can be performed usinglow fragment-to-bead ratios (N/M<1). This
minimizes the number of beads associated with more than one DNA. A
separation step can be used to remove most or all of the beads with
no DNA, leaving an enriched population of beads with one or more
species of amplified DNA. This enriched population may be analyzed
by any method of sequencing such as, for example, pyrophosphate
sequencing. Because the fraction of beads with one amplicon (N=1)
is enriched, any method of sequencing can be applied more
efficiently.
As an example, with an average fragment-to-bead ratio of 0.1, 90%
of the beads will carry no amplicon, 9% of the beads will carry one
amplicon, and 0.5% of the beads will carry more than one amplicon.
The enrichment described herein below will remove the 90% of the
zero amplicon beads leaving a population of beads where the
fraction available for sequencing (N=1) is: 1-(0.005/0.09)=94%.
Dilution of the fragment to bead mixture, along with separation of
beads containing amplicon can yield an enrichment of 2.5 fold over
the optimal unenriched method. For example, 94%/37% (See Table 2,
above, N/M=1)=2.5. An additional benefit of the enrichment
procedure described herein below is that the ultimate fraction of
beads useful for sequencing is relatively insensitive to
variability in N/M. Thus, complex calculations to derive the
optimal N/M ratio are either unnecessary or may be performed with
lower levels of precision. Accordingly, the methods of the
invention can be easily adapted for use by less trained personnel
or automation. An additional benefit of these methods is that the
zero amplicon beads may be recycled and reused. While recycling is
not necessary, it may reduce cost or the total bulk of reagents
making the method of the invention more suitable for some purposes
such as, for example, portable sampling, remote robotic sampling,
and the like. In addition, the collective benefits of the disclosed
methods (e.g., adaptation for less trained personnel, automation,
and recycling of reagents) will reduce the costs of the methods.
The enrichment procedure is described in more detail below.
The enrichment procedure may be used to treat beads that have been
amplified in the bead emulsion method described above. The
amplification is designed so that each amplified nucleic acid
molecule contains the same sequence at its 3' end. The nucleotide
sequence may be a 20 mer but may be any sequence from 15 bases or
more such as 25 bases, 30 bases, 35 bases, 40 bases, or longer.
While longer oligonucleotide ends are functional, they are not
necessary. This 3' sequence may be introduced at the end of an
amplified nucleic acid by one of skill in the art. For example, if
PCR is used for amplification of a DNA template, the sequence may
be included as part of one member of the PCR primer pair.
A schematic of the enrichment process is depicted in FIG. 3. In
this process, the amplicon-bound bead is mixed with four empty
beads to create a fragment-diluted amplification bead mixture. In
step 1, a biotinylated primer complementary to the 3' end of the
amplicon is annealed to the amplicon. In step 2, DNA polymerase and
the four natural deoxynucleotide triphosphates (dNTPs) are added to
the bead mixture and the biotinylated primer is extended. This
extension is to enhance the bonding between the biotinylated primer
and the bead-bound DNA. This step may be omitted if the
biotinylated primer--DNA bond is strong (e.g., in a high ionic
environment). In Step 3, streptavidin coated beads susceptible to
attraction by a magnetic field (referred to herein as "magnetic
streptavidin beads") are introduced to the bead mixtures. Magnetic
beads are commercially available, for example, from Dynal (M290).
The streptavidin capture moieties binds biotin groups hybridized to
the amplicons, thereby binding the amplicon-bound beads to the
magnetic streptavidin beads.
In step 5, a magnetic field (represented by a magnet) is applied
near the reaction mixture, which causes the magnetic streptavidin
beads/amplicon bound bead complexes to be positioned along one side
of the tube most proximal to the magnetic field. Magnetic beads
without amplicon bound beads attached are also expected to be
positioned along the same side. Beads without amplicons remain in
solution. The bead mixture is washed and the beads not bound by the
magnet (i.e., the empty beads) are removed and discarded. In step
6, the extended biotinylated primer strand is separated from the
amplicon strand by "melting." This step that can be accomplished,
for example, by heat or a change in pH. The heat may be 60.degree.
C. in low salt conditions (e.g., in a low ionic environment such as
0.1.times.SSC). The change in pH may be accomplished by the
addition of NaOH. Next, the mixture is washed and the supernatant
containing the amplicon bound beads is recovered, while the
magnetic beads are retained by a magnetic field. The resultant
enriched beads may be used for DNA sequencing. It is noted that the
primer on the DNA capture bead may be the same as the primer of
step 2, above. In this case, annealing of the amplicon-primer
complementary strands (with or without extension) is the source of
target-capture affinity.
The biotin streptavidin pair could be replaced by a variety of
capture-target pairs. For example, capture-target pairs can employ
reversible (e.g., cleavable) or irreversible linkages. Non-limiting
examples of reversible linkages include thiol-thiol,
digoxigenin/anti-digoxigenin, and linkages using VECTREX.RTM.
Avidin DLA (Vector Laboratories, Burlingame, Calif.),
CaptAvidin.TM., NeutrAvidin.TM., and D-desthiobiotin (Molecular
Probes, Inc., Eugene, Oreg.).
As described above, step 2 of the enrichment process is optional.
If step 2 is omitted, it may not be necessary to separate the
magnetic beads from the amplicon bound beads. The amplicon bound
beads, with the magnetic beads attached, may be used directly for
sequencing. For example, separation may not be necessary if
sequencing is to be performed in a microtiter or picotiter plate
and the amplicon bound bead-magnetic bead complex can fit inside
the well of the plate.
While the use of magnetic capture beads is convenient, capture
moieties can encompass other binding surfaces. For example,
streptavidin can be chemically bound to a surface such as the inner
surface of a tube. In this case, the amplified bead mixture may be
flowed through the tube. The amplicon bound beads will tend to be
retained until "melting" while the empty beads will flow through.
This arrangement may be particularly advantageous for automating
the bead preparation process.
While the embodiments described above are particularly useful,
other methods to separate beads can be envisioned. For example, the
capture beads may be labeled with a fluorescent moiety which would
make the target-capture bead complex fluorescent. The target
capture bead complex may be separated by flow cytometry or
fluorescence cell sorter. Using large capture beads would allow
separation by filtering or other particle size separation
techniques. Since both capture and target beads are capable of
forming complexes with a number of other beads, it is possible to
agglutinate a mass of cross-linked capture-target beads. The large
size of the agglutinated mass would make separation possible by
simply washing away the unagglutinated empty beads. These methods
described are described in more detail, for example, in Bauer, J.;
J. Chromatography B, 722 (1999) 55-69 and in Brody et al., Applied
Physics Lett. 74 (1999) 144-146.
In one embodiment, the invention encompasses a method for
amplifying one or more nucleic acids comprising the steps of: a)
forming a water-in-oil emulsion to create a plurality of aqueous
microreactors wherein at least one of the microreactors comprises a
single nucleic acid template, a single bead capable of binding to
the nucleic acid, and amplification reaction solution containing
reagents necessary to perform nucleic acid amplification; b)
amplifying the nucleic acids in the microreactors to form amplified
copies of the nucleic acids; and c) binding the amplified copies to
the beads in the microreactors.
The amplification reaction solution used with this method may be a
polymerase chain reaction solution comprising nucleotide
triphosphates, a thermostable polymerase, and nucleic acid primers
suspended in a buffer compatible with polymerase chain reaction
conditions. The polymerase chain reaction is may be an asymmetric
polymerase chain reaction or a symmetric polymerase chain reaction.
As examples, amplification may be carried out by
transcription-based amplification, rapid amplification of cDNA
ends, continuous flow amplification, or rolling circle
amplification.
For use with this method, a majority of the microreactors may
include a single nucleic acid. The method may be performed with at
least 10,000 nucleic acids, or at least 50,000 nucleic acids. Each
bead used with the method can be used to capture more than 10,000
amplification copies of a nucleic acid template. In various
embodiments, the emulsion additionally contains emulsion
stabilizers. The emulsion stabilizers may be Atlox 4912, Span 80,
or combinations or mixtures thereof. The emulsion may be heat
stable, e.g., to 95.degree. C., and may be formed by the dropwise
addition of the nucleic acid templates, beads, and amplification
reaction solution into an oil. The microreactors may have an
average size of 50 to 250 .mu.m in diameter.
In another embodiment, the invention encompasses a library
comprising a plurality of nucleic acid molecules, wherein each
nucleic acid molecule is separately immobilized to a different
bead, and wherein each bead comprises over 1,000,000 clonal
amplification copies of each nucleic acid molecule, wherein the
library is contained in a single vessel. As examples, the nucleic
acid molecules may be genomic DNA, cDNA, episomal DNA, BAC DNA, or
YAC DNA. The genomic DNA may be animal, plant, viral, bacterial, or
fungal genomic DNA. Preferably, the genomic DNA is human genomic
DNA or human cDNA. In certain aspects, the bead, e.g., a Sepharose
bead, has a diameter of 2 microns to 100 microns.
The invention also encompasses a method for amplifying a nucleic
acid comprising the steps of: a) providing a nucleic acid template
to be amplified; b) providing a solid support material comprising a
generally spherical bead having a diameter about 10 to about 80 m,
wherein the bead is capable of binding to the nucleic acid
template; c) mixing the nucleic acid template and the bead in an
amplification reaction solution containing reagents necessary to
perform a nucleic acid amplification reaction in a water-in-oil
emulsion; d) amplifying the nucleic acid template to form amplified
copies of the nucleic acid template; and e) binding the amplified
copies to the bead.
As an option, the method can include an enrichment step to isolate
beads which bind amplified copies of the nucleic acid away from
beads to which no nucleic acid is bound. This enrichment step may
be performed by electrophoresis, cell sorting, or affinity
purification (e.g., with magnetic beads that bind nucleic acid).
Preferably, at least 100,000 copies of each target nucleic acid
molecule are bound to each bead, at least 1,000,000 copies of each
target nucleic acid molecule are bound to each bead, or at least 1
to 20,000,000 copies of each target nucleic acid molecule are bound
to each bead. In various aspects, the beads are Sepharose beads and
amplified copies are bound to the beads by a binding pair such as
antigen/antibody, ligand/receptor, polyhistidine/nickel, or
avidin/biotin. The method can also include the steps of: f)
separating the template carrying beads and magnetic bead; and g)
removing the magnetic beads with a magnetic field. This separation
may be achieved by incubation at a temperature greater than
45.degree. C. or by incubating the template carrying beads and the
magnetic beads in a solution with a basic pH.
The invention further encompasses a kit for conducting nucleic acid
amplification of a nucleic acid template comprising: a) a nucleic
acid capture bead; b) an emulsion oil; c) one or more emulsion
stabilizers; and d) instructions for employing the kit.
Additionally, the invention encompasses a method for producing a
clonal population of nucleic acids, comprising: a) providing a
plurality of nucleic acid templates from 50-800 bp in length and
beads capable of binding to the nucleic acid templates; b) mixing
the nucleic acid templates and the beads in a biological reaction
solution containing reagents necessary to amplify the nucleic acid
templates; and c) forming an emulsion to create a plurality of
microreactors comprising the nucleic acid templates, beads, and
biological reaction solution, wherein at least one of the
microreactors comprises a single nucleic acid template and a single
bead encapsulated in the biological reaction solution, wherein the
microreactors are contained in the same vessel.
In accordance with this method, the nucleic acids can be
transcribed and translated to generate at least 10,000 copies of an
expression product. The expression product may be bound to the
beads by a binding pair selected from the group consisting of
antigen/antibody, ligand/receptor,
6.times.his/nickel-nitrilotriacetic acid, and FLAG tag/FLAG
antibody binding pairs. In certain aspects, the method produces a
clonal population of proteins, such as antibodies, antibodies
fragments, and engineered antibodies. The emulsion may comprise a
plurality of thermostable microreactors, wherein the microreactors
are 50 to 200 .mu.m in diameter and comprise a biological reaction
solution. The biological reaction solution may comprise reagents
for performing polymerase chain reaction amplification reactions or
coupled transcription and translation reactions. Preferably, a
plurality of microreactors comprise a nucleic acid template, e.g.,
one or fewer nucleic acid templates, and one or fewer beads that
bind to the nucleic acid templates.
EXAMPLES
Beam Emulsion PCR
The following procedures, including capture of the template DNA,
DNA amplification, and recovery of the beads bound to amplified
template, can be performed in a single tube. The emulsion format
ensures the physical separation of the beads into 100-200 .mu.m
"microreactors" within this single tube, thus allowing for clonal
amplification of the various templates. Immobilization of the
amplification product is achieved through extension of the template
along the oligonucleotides hound to the DNA capture beads. Typical,
the copy number of the immobilized template ranges from 10 to 30
million copies per bead. The DNA capture beads affixed with
multiple copies of a single species of nucleic acid template are
ready for distribution onto PTPs.
The 300,000 75-picoliter wells etched in the PTP surface provide a
unique array for the sequencing of short DNA templates in a
massively parallel, efficient and cost-effective manner. However,
this requires fairly large quantities (millions of copies) of
clonal templates in each reaction well. The methods of the
invention allow the user to clonally amplify single-stranded
genomic template species thorough PCR reactions conducted in
standard tubes or microliter plates. Single copies of the template
species may be mixed with capture beads, resuspended into complete
PCR amplification solution, and emulsified into microreactors (100
to 200 .mu.M in diameter), after which PCR amplification generates
10.sup.7-fold amplification of the initial template species. This
procedure is much simpler and more cost-effective than previous
methods.
Example 1
Binding Nucleic Acid Template to Capture Beads
This example describes preparation of a population of beads that
preferably have only one unique nucleic acid template attached
thereto. Successful clonal amplification depends on the delivery of
a controlled number of template species (0.5 to 1) to each bead.
Delivery of excess species can result in PCR amplification of a
mixed template population, preventing generation of meaningful
sequence data While a deficiency of species will result in fewer
wells containing template for sequencing. This can reduce the
extent of genome coverage provided by the sequencing phase. As a
result, it is preferred that the template concentration be
accurately determined through replicated quantitation, and that the
binding protocol be followed as outlined below.
Template Quality Control
The success of the Emulsion PCR reaction is related to the quality
of the template species. Regardless of the care and detail paid to
the amplification phase, poor quality templates will impede
successful amplification and the generation of meaningful sequence
data. To prevent unnecessary loss of time and money, it is
important to check the quality of the template material before
initiating the Emulsion PCR phase of the process. Preferably, the
library should pass two quality control steps before it is used in
Emulsion PCR. Its concentration and the distribution of products it
contains should be determined. Ideally, the library should appear
as a heterogeneous population of fragments with little or no
visible adapter dimers (e.g., .about.90 bases). Also, amplification
with PCR primers should result in a product smear ranging, for
example, from 300 to 500 bp. Absence of amplification product may
reflect failure to properly ligate the adaptors to the template,
while the presence of a single band of any size may reflect
contamination of the template.
Preparation of the PCR Solution
The main consideration for this phase is to prevent contamination
of the PCR reaction mixture with stray amplicons. Contamination of
the PCR reactions with a residual amplicon is one of the critical
issues that can cause failure of a sequencing nm. To reduce the
possibility of contamination, proper lab technique should be
followed, and reaction mixture preparation should be conducted in a
clean room in a UV-treated laminar flow hood.
PCR Reaction Mix:
For 200 .mu.l PCR reaction mixture (enough for amplifying 600,000
beads), the following reagents were combined in a 0.2 ml PCR
tube:
TABLE-US-00003 TABLE 3 Stock Final Microliters HIFI Buffer
10.times. 1.times. 20 treated nucleotides 10 mM 1 mM 20 Mg 50 mM 2
mM 8 BSA 10% 0.1% 2 Tween 80 1% 0.01% 2 Ppase 2 U 0.003 U 0.333333
Primer MMP1a 100 .mu.M 0.625 .mu.M 1.25 Printer MMP1b 10 .mu.M
0.078 .mu.M 1.56 Taq polymerase 5 U 0.2 U 8 Water 136.6 Total
200
The tube was vortexed thoroughly and stored on ice until the beads
are annealed with template.
DNA Capture Beads:
1. 600,000 DNA capture beads were transferred from the stock tube
to a 1.5 ml microfuge tube. The exact amount used will depend on
bead concentration of formalized reagent.
2. The beads were pelleted in a benchtop mini centrifuge and
supernatant was removed.
3. Steps 4-11 were performed in a PCR Clean Room.
4. The beads were washed with 1 mL of 1.times. Annealing
Buffer.
5. The capture beads were pelleted in the microcentrifuge. The tube
was turned 180.degree. and spun again.
6. All but approximately 10 .mu.l of the supernatant was removed
from the tube containing the beads. The beads were not
disturbed.
7. 1 mL of 1.times. Annealing Buffer was added and this mixture was
incubated for 1 minute. The beads were then pelleted as in step
5.
8. All but approximately 100 .mu.L of the material from the tube
was removed.
9. The remaining beads and solution were transferred to a PCR
tube.
10. The 1.5 mL tube was washed with 150 .mu.L of 1.times. Annealing
Buffer by pipetting up and down several times. This was added to
the PCR tube containing the beads.
11. The beads were pelleted as in step 5 and all but 1.0 .mu.L of
supernatant was removed, taking care to not disturb the bead
pellet.
12. An aliquot of quantitated single-stranded template DNA (sstDNA)
was removed. The final concentration was 200,000-sst DNA
molecules/.mu.l.
13. 3 .mu.l of the diluted sstDNA was added to PCR tube containing
the beads. This was equivalent to 600,000 copies of sstDNA.
14. The tube was vortexed gently to mix contents.
15. The sstDNA was annealed to the capture beads in a PCR
thermocycler with the program 80 Anneal stored in the EPCR folder
on the MJ Thermocycler, using the following protocol: 5 minutes at
65.degree. C.; Decrease by 0.1.degree. C./sec to 60.degree. C.;
Hold at 60.degree. C. for 1 minute; Decrease by 0.1.degree. C./sec
to 50.degree. C.; Hold at 50.degree. C. for 1 minute; Decrease by
0.1.degree. C./sec to 40.degree. C.; Hold at 40.degree. C. for 1
minute; Decrease by 0.1.degree. C./sec to 20.degree. C.; and Hold
at 10.degree. C. until ready for next step.
In most cases, beads were used for amplification immediately after
template binding. If beads were not used immediately, they should
were stored in the template solution at 4.degree. C. until needed.
After storage, the beads were treated as follows.
16. As in step 6, the beads were removed from the thermocycler,
centrifuged, and annealing buffer was removed without disturbing
the beads.
17. The beads were stored in an ice bucket until emulsification
(Example 2).
18. The capture beads included, on average, 0.5 to 1 copies of
sstDNA bound to each bead, and were ready for emulsification.
Example 2
Emulsification
This example describes how to create a heat-stable water-in-oil
emulsion containing about 3,000 PCR microreactors per microliter.
Outlined below is a protocol for preparing the emulsion.
1. 200 .mu.l of PCR solution was added to the 600,000 beads (both
components from Example 1).
2. The solution was pipetted up and down several times to resuspend
the beads.
3. The PCR-bead mixture was allowed to incubate at room temperature
for 2 minutes to equilibrate the beads with PCR solution.
4. 400 .mu.l of Emulsion Oil was added to a UV-irradiated 2 ml
microfuge tube.
5. An "amplicon-free" 1/4'' stir magnetic stir bar was added to the
tube of Emulsion Oil.
An amplicon-free stir bar was prepared as follows. A large stir bar
was used to hold a 1/4'' stir bar. The stir bar was then: Washed
with DNA-Off (drip or spray); Rinsed with picopure water; Dried
with a Kimwipe edge; and UV irradiated for 5 minutes.
6. The magnetic insert of a Dynal MPC-S tube holder was removed.
The tube of Emulsion Oil was placed in the tube holder. The tube
was set in the center of a stir plate set at 600 rpm.
7. The tube was vortexed extensively to resuspend the beads. This
ensured that there was minimal clumping of beads.
8. Using a P-200 pipette, the PCR-bead mixture was added drop-wise
to the spinning oil at a rate of about one drop every 2 seconds,
allowing each drop to sink to the level of the magnetic stir bar
and become emulsified before adding the next drop. The solution
turned into a homogeneous milky white liquid with a viscosity
similar to mayonnaise.
9. Once the entire PCR-bead mixture was been added, the micronize
tube was flicked a few times to mix any oil at the surface with the
milky emulsion.
10. Stirring was continued for another 5 minutes.
11. Steps 9 and 10 were repeated.
12. The stir bar was removed from the emulsified material by
dragging it out of the tube with a larger stir bar.
13. 10 .mu.L of the emulsion was removed and placed on a microscope
slide. The emulsion was covered with a cover slip and the emulsion
was inspected at 50.times. magnification (10.times. ocular and
5.times. objective lens). A "good" emulsion was expected to include
primarily single beads in isolated droplets (microreactors) of PCR
solution in oil.
14. A suitable emulsion oil mixture with emulsion stabilizers was
made as follows. The components for the emulsion mixture are shown
in Table 4.
TABLE-US-00004 TABLE 4 Quantity Ingredient Required Source Ref.
Number Sigma Light Mineral Oil 94.5 g Sigma M-5904 Atlox 4912 1 g
Uniqema NA Span 80 4.5 g Uniqema NA
The emulsion oil mixture was made by prewarming the Atiox 4912 to
60.degree. C. in a water bath. Then, 4.5 grams of Span 80 was added
to 94.5 grams of mineral oil to form a mixture. Then, one gram of
the prewarmed Adox 4912 was added to the mixture. The solutions
were placed in a closed container and mixed by shaking and
inversion. Any sign that the Atiox was settling or solidifying was
remedied by warming the mixture to 60.degree. C., followed by
additional shaking.
Example 3
Amplification
This example describes amplification of the template DNA in the
bead emulsion mixture. According to this protocol of the invention,
the DNA amplification phase of the processtakes 3 to 4 hours. After
the amplification is complete, the emulsion may be left on the
thermocycler for up to 12 hours before beginning the process of
isolating the beads. PCR thermocycling was performed by placing 50
to 100 .mu.l of the emulsified reaction mixture into individual PCR
reaction chambers (i.e., PCR tubes). PCR was performed as
follows:
1. The emulsion was transferred in 50-100 .mu.L amounts into
approximately 10 separate PCR tubes or a 96-well plate using a
single pipette tip. For this step, the water-in-oil emulsion was
highly viscous.
2. The plate was sealed, or the PCR tube lids were closed, and the
containers were placed into in a MJ thermocycler with or without a
96-well plate adaptor.
3. The PCR thermocycler was programmed to run the following
program: 1 cycle (4 minutes at 94.degree. C.)--Hotstart Initiation;
40 cycles (30 seconds at 94.degree. C., 30 seconds at 58.degree.
C., 90 seconds at 68.degree. C.); 25 cycles (30 seconds at
94.degree. C., 6 minutes at 58.degree. C.); and Storage at
14.degree. C.
4. After completion of the PCR reaction, the amplified material was
removed in order to proceed with breaking the emulsion and bead
recovery.
Example 4
Breaking the Emulsion and Bead Recovery
This example describes how to break the emulsion and recover the
beads with amplified template thereon. Preferably, the post-PCR
emulsion should remain intact. The lower phase of the emulsion
should, by visual inspection, remain a milky white suspension. If
the solution is clear, the emulsion may have partially resolved
into its aqueous and oil phases, and it is likely that many of the
beads will have a mixture of templates. If the emulsion has broken
in one or two of the tubes, these samples should not be combined
with the others. If the emulsion has broken in all of the tubes,
the procedure should not be continued.
1. All PCR reactions from the original 600 .mu.l sample were
combined into a single 1.5 ml microfuge tube using a single pipette
tip. As indicated above, the emulsion was quite viscous. In some
cases, pipetting was repeated several times for each tube. As much
material as possible was transferred to the 1.5 ml tube.
2. The remaining emulsified material was recovered from each PCR
tube by adding 50 .mu.l of Sigma Mineral Oil into each sample.
Using a single pipette tip, each tube was pipetted up and down a
few times to resuspend the remaining material.
3. This material was added to the 1.5 ml tube containing the bulk
of the emulsified material.
4. The sample was vortexed for 30 seconds.
5. The sample was spun for 20 minutes in the tabletop microfuge
tube at 13.2K rpm in the Eppendorf microcentrifuge.
6. The emulsion separated into two phases with a large white
interface. As much of the top, clear oil phase as possible was
removed. The cloudy material was left in the tube. Often a white
layer separated the oil and aqueous layers. Beads were often
observed pelleted at the bottom of the tube.
7. The aqueous layer above the beads was removed and saved for
analysis (gel analysisAgilent 2100, and Taqman). If an interface of
white material persisted above the aqueous layer, 20 microliters of
the underlying aqueous layer was removed. This was performed by
penetrating the interface material with a pipette tip and
withdrawing the solution from underneath.
8. In the PTP Fabrication and Surface Chemistry Room Fume Hood, 1
ml of Hexanes was added to the remainder of the emulsion.
9. The sample was vortexed for 1 minute and spun at full speed for
1 minute.
10. In the PTP Fabrication and Surface Chemistry Room Fume Hood,
the top, oil/hexane phase was removed and placed into the organic
waste container.
11. 1 ml of 1.times. Annealing Buffer was added in 80% Ethanol to
the remaining aqueous phase, interface, and beads.
12. The sample was vortexed for 1 minute or until the white
substance dissolved.
13. The sample was centrifuged for 1 minute at high speed. The tube
was rotated 180 degrees, and spun again for 1 minute. The
supernatant was removed without disturbing the bead pellet.
14. The beads were washed with 1 ml of 1.times. Annealing Buffer
containing 0.1% Tween 20 and this step was repeated.
Example 5
Single Strand Removal and Primer Annealing
If the beads are to be used in a pyrophosphate-based sequencing
reaction, then it is necessary to remove the second strand of the
PCR product and anneal a sequencing primer to the single stranded
template that is bound to the bead. This example describes a
protocol for accomplishing that.
1. The beads were washed with 1 ml of water, and spun twice for 1
minute. The tube was rotated 180.degree. between spins. After
spinning, the aqueous phase was removed.
2. The beads were washed with 1 ml of 1 mM EDTA. The tube was spun
as in step 1 and the aqueous phase was removed.
3. 1 ml of 0.125 M NaOH was added and the sample was incubated for
8 minutes.
4. The sample was vortexed briefly and placed in a
microcentrifuge.
5. After 6 minutes, the beads were pelleted as in step 1 and as
much solution as possible was removed.
6. At the completion of the 8 minute NaOH incubation, 1 ml of
1.times. Annealing Buffer was added.
7. The sample was briefly vortexed, and the beads were pelleted as
in step 1. As much superiatant as possible was removed, and another
1 ml of 1.times. Annealing buffer was added.
8. The sample was briefly vortexed, the beads were pelleted as in
step 1, and 800 .mu.l of 1.times. Annealing Buffer was removed.
9. The beads were transferred to a 0.2 ml PCR tube.
10. The beads were transferred and as much Annealing Buffer as
possible was removed, without disturbing the beads.
11. 100 .mu.l of 1.times. Annealing Buffer was added.
12. 4 .mu.l of 100 .mu.M sequencing primer was added. The sample
was vortexed just prior to annealing.
13. Annealing was performed in a MJ thermocycler using the
"80Anneal" program.
14. The beads were washed three times with 200 .mu.l of 1.times.
Annealing Buffer and resuspended with 100 of 1.times. Annealing
Buffer.
15. The beads were counted in a Hausser Hemacytometer. Typically,
300,000 to 500,000 beads were recovered (3,000-5,000
beads/.mu.L).
16. Beads were stored at 4.degree. C. and could be used for
sequencing for 1 week.
Example 6
Optional Enrichment Step
The beads may be enriched for amplicon containing bead using the
following procedure. Enrichment is not necessary but it could be
used to make subsequent molecular biology techniques, such as DNA
sequencing, more efficient.
Fifty microliters of 10 .mu.M (total 500 pmoles) of
biotin-sequencing primer was added to the Sepharose beads
containing amplicons from Example 5. The beads were placed. In a
thermocycler. The primer was annealed to the DNA on the bead by the
thermocycler annealing program of Example 2.
After annealing, the sepharose beads were washed three times with
Annealing Buffer containing 0.1% Tween 20. The beads, now
containing ssDNA fragments annealed with biotin-sequencing primers,
were concentrated by centrifugation and resuspended in 200 .mu.l of
BST binding buffer. Ten microliters of 50,000 unit/mi
Bst-polymerase was added to the resuspended beads and the vessel
holding the beads was placed on a rotator for five minutes. Two
microliters of 10 mM dNTP mixture (i.e., 2.5 .mu.l each of 10 mM
dATP, dGTP, dCTP and dTTP) was added and the mixture was incubated
for an additional 10 minutes at room temperature. The beads were
washed three times with annealing buffer containing 0.1% Tween 20
and resuspended in the original volume of annealing buffer.
Fifty microliters of Dynal Streptavidin beads (Dynal Biotech Inc.,
Lake Success, N.Y.; M270 or MyOne.TM. beads at 10 mg/ml) was washed
three times with Annealing Buffer containing 0.1.% Tween 20 and
resuspended in the original volume in Annealing Buffer containing
0.1% Tween 20. Then the Dynal bead mixture was added to the
resuspended sepharose beads. The mixture was vortexed and placed in
a rotator for 10 minutes at room temperature.
The beads were collected on the bottom of the test tube by
centrifugation at 2300 g (500 rpm for Eppendorf Centrifuge 5415D).
The beads were resuspended in the original volume of Annealing
Buffer containing 0.1% Tween 20. The mixture, in a test tube, was
placed in a magnetic separator (Dynal). The beads were washed three
times with Annealing Buffer containing 0.1% Tween 20 and
resuspended in the original volume in the same buffer. The beads
without amplicons were removed by wash steps, as previously
described. Only Sepharose beads containing the appropriated DNA
fragments were retained.
The magnetic beads were separated from the sepharose beads by
addition of 500 .mu.l of 0.125 M NaOH. The mixture was vortexed and
the magnetic beads were removed by magnetic separation. The
Sepharose beads remaining in solution was transferred to another
tube and washed with 400 .mu.l of 50 mM Tris Acetate until the pH
was stabilized at 7.6.
Example 7
Nucleic Acid Sequencing Using Bead Emulsion PCR
The following experiment was performed to test the efficacy of the
bead emulsion PCR. For this protocol, 600,000 Sepharose beads, with
an average diameter of 25-35 .mu.m (as supplied my the
manufacturer) were covalently attached to capture primers at a
ratio of 30-50 million copies per bead. The beads with covalently
attached capture primers were mixed with 1.2 million copies of
single stranded Adenovirus Library. The library constructs included
a sequence that was complimentary to the capture primer on the
beads.
The adenovirus library was annealed to the beads using the
procedure described in Example 1. Then, the beads were resuspended
in complete PCR solution. The PCR Solution and beads were
emulsified in 2 volumes of spinning emulsification oil using the
same procedure described in Example 2. The emulsified
(encapsulated) beads were subjected to amplification by PCR, as
outlined in Example 3. The emulsion was broken as outlined in
Example 4. DNA on beads was rendered single stranded, sequencing
primer was annealed using the procedure of Example 5.
Next, 70,000 beads were sequenced simultaneously by pyrophosphate
sequencing using a pyrophosphate sequencer from 454 Life Sciences
(New Haven, Conn.) (see co-pending application of Lohman et al.,
filed concurrently herewith entitled "Methods of Amplifying and
Sequencing Nucleic Acids" U.S. Ser. No. 60/476,592 filed Jun. 6,
2003). Multiple batches of 70,000 beads were sequenced and the data
were listed in Table 5, below.
TABLE-US-00005 TABLE 5 Alignment Inferred Error Alignments Read
Tolerance None Single Multiple Unique Coverage Error 0% 47916 1560
1110 54.98% 0.60% 5% 46026 3450 2357 83.16% 1.88% 10% 43474 6001 1
3742 95.64% 4.36%
Table 5 shows the results obtained from BLAST analysis comparing
the sequences obtained from the pyrophosphate sequencer against
Adenovirus sequence. The first column shows the error tolerance
used in the BLAST program. The last column shows the real error as
determined by direct comparison to the known sequence.
Bead Emulsion PCR for Double Ended Sequencing
Example 8
Template Quality Control
As indicated previously, the success of the Emulsion PCR reaction
was found to be related to the quality of the single stranded
template species. Accordingly, the quality of the template material
was assessed with two separate quality controls before initiating
the Emulsion PCR protocol. First, an aliquot of the single-stranded
template was run on the 2100 BioAnalyzer (Agilient). An RNA Pico
Chip was used to verify that the sample included a heterogeneous
population of fragments, ranging in size from approximately 200 to
500 bases. Second, the library was quantitated using the RiboGreen
fluorescence assay on a Bio-Tek FL600 plate fluorometer. Samples
determined to have DNA concentrations below 5 ng/.mu.l were deemed
too dilute for use.
Example 9
DNA Capture Bead Synthesis
Packed beads from a 1 mL N-hydroxysuccinimide ester (NHS)-activated
Sepharose HP affinity column (Amersham Biosciences, Piscataway,
N.J.) were removed from the column. The 30-25 .mu.m size beads were
selected by serial passage through 30 and 25 .mu.m pore filter mesh
sections (Sefar America, Depew, N.Y., USA). Beads that passed
through the first filter, but were retained by the second were
collected and activated as described in the product literature
(Amersham Pharmacia Protocol #71700600AP). Two different
amine-labeled HEG (hexaethyleneglycol) long capture primers were
obtained, corresponding to the 5' end of the sense and antisense
strand of the template to be amplified, (5'-Amine-3 HEG spacers
gcttacctgaccgacctctgcctatcccctgttgcgtgtc-3'); SEQ ID NO:1; and
5'-Amine-3 HEG spacers ccattccccagctcgtcttgccatctgttccctccctgtc-3';
SEQ ID NO:2) (IDT Technologies, Coralville, Iowa, USA). The primers
were designed to capture of both strands of the amplification
products to allow double ended sequencing, i.e., sequencing the
first and second strands of the amplification products. The capture
primers were dissolved in 20 mM phosphate buffer, pH 8.0, to obtain
a final concentration of 1 mM. Three microliters of each primer
were bound to the sieved 30-25 .mu.m beads. The beads were then
stored in a bead storage buffer (50 mM Tris, 0.02% Tween and 0.02%
sodium azide, pH 8). The beads were quantitated with a
hemacytometer (Hausser Scientific, Horsham, Pa., USA) and stored at
4.degree. C. until needed.
Example 10
PCR Reaction Mix Preparation and Formulation
As with any single molecule amplification technique, contamination
of the reactions with foreign or residual amplicon from other
experiments could interfere with a sequencing run. To reduce the
possibility of contamination, the PCR reaction mix was prepared in
a in a UV-treated laminar flow hood located in a PCR clean room.
For each 600,000 bead emulsion PCR reaction, the following reagents
were mixed in a 1.5 ml tube: 225 .mu.l of reaction mixture
(1.times. Platinum HiFi Buffer (Invitrogen)), 1 .mu.M dNTPs, 2.5 mM
MgSO.sub.4 (Invitrogen), 0.1% BSA, 0.01% Tween, 0.003 thermostable
PPi-ase (NEB), 0.125 .mu.M forward primer
(5'-gettacctgaccgacctetg-3'; SEQ ID NO:3) and 0.125 .mu.M reverse
primer (5'-ccattccccagctcgtcttg-3'; SEQ ID NO:4) (IDT Technologies,
Coralville, Iowa, USA) and 0.2 U/.mu.l Platinum HiFi Taq Polymerase
(Invitrogen). Twenty-five microliters of the reaction mixture was
removed and stored in an individual 200 .mu.l PCR tube for use as a
negative control. Both the reaction mixture and negative controls
were stored on ice until needed.
Example 11
Binding Template Species to DNA Capture Beads
Successful clonal DNA amplification for sequencing relates to the
delivery of a controlled number of template species to each bead.
For the experiments described herein below, the typical target
template concentration was determined to be 0.5 template copies per
capture bead. At this concentration, Poisson distribution dictates
that 61% of the beads have no associated template, 30% have one
species of template, and 9% have two or more template species.
Delivery of excess species can result in the binding and subsequent
amplification of a mixed population (2 or more species) on a single
bead, preventing the generation of meaningful sequence data.
However, delivery of too few species will result in fewer wells
containing template (one species per bead), reducing the extent of
sequencing coverage. Consequently, it was deemed that the
single-stranded library template concentration was important.
Template nucleic acid molecules were annealed to complimentary
primers on the DNA capture beads by the following method, conducted
in a UV-treated laminar flow hood. Six hundred thousand. DNA
capture beads suspended in bead storage buffer (see Example 9,
above) were transferred to a 200 .mu.l PCR tube. The tube was
centrifuged in a benchtop mini centrifuge for 10 seconds, rotated
180.degree., and spun for an additional 10 seconds to ensure even
pellet formation. The supernatant was removed, and the beads were
washed with 200 .mu.l of Annealing Buffer (20 mM Tris, pH 7.5 and 5
mM magnesium acetate). The tube was vortexed for 5 seconds to
resuspend the beads, and the beads were pelleted as before. All but
approximately 10 .mu.l of the supernatant above the beads was
removed, and an additional 200 .mu.l of Annealing Buffer was added.
The beads were again vortexed for 5 seconds, allowed to sit for 1
minute, and then pelleted as before. All but 10 .mu.l of
supernatant was discarded.
Next, 1.5 .mu.l of 300,000 molecules/.mu.l template library was
added to the beads. The tube was vortexed for 5 seconds to mix the
contents, and the templates were annealed to the beads in a
controlled denaturation/annealing program preformed in an MJ
thermocycler. The program allowed incubation for 5 minutes at
80.degree. C., followed by a decrease by 0.1.degree. C./sec to
70.degree. C., incubation for 1 minute at 70.degree. C., decrease
by 0.1.degree. C./sec to 60.degree. C., hold at 60.degree. C. for 1
minute, decrease by 0.1.degree. C./sec to 50.degree. C., hold at
50.degree. C. for 1 minute, decrease by 0.1.degree. C./sec to
20.degree. C., hold at 20.degree. C. Following completion of the
annealing process, the beads were removed from the thermocycler,
centrifuged as before, and the Annealing Buffer was carefully
decanted. The capture beads included on average 0.5 copy of single
stranded template DNA bound to each bead, and were stored on ice
until needed.
Example 12
Emulsification
The emulsification process creates a heat-stable water-in-oil
emulsion containing 10,000 discrete PCR microreactors per
microliter. This serves as a matrix for single molecule, clonal
amplification of the individual molecules of the target library.
The reaction mixture and DNA capture beads for a single reaction
were emulsified in the following manner. In a UV-treated laminar
flow hood, 200 .mu.l of PCR solution (from Example 10) was added to
the tube containing the 600,000 DNA capture beads (from Example
11). The beads were resuspended through repeated pipetting. After
this, the PCR-bead mixture was incubated at room temperature for at
least 2 minutes, allowing the beads to equilibrate with the PCR
solution. At the same time, 450 .mu.l of Emulsion Oil (4.5% (w:w)
Span 80, 1% (w:w) Atlox 4912 (Uniqema, Del.) in light mineral oil
(Sigma)) was aliquotted into a flat-topped 2 ml centrifuge tube
(Dot Scientific) containing a sterile 1/4 inch magnetic stir bar
(Fischer). This tube was then placed in a custom-made plastic tube
holding jig, which was then centered on a Fisher Isotemp digital
stilling hotplate (Fisher Scientific) set to 450 RPM.
The PCR-bead solution was vortexed for 15 seconds to resuspend the
beads. The solution was then drawn into a 1 ml disposable plastic
syringe (Benton-Dickenson) affixed with a plastic safety syringe
needle (Henry Schein). The syringe was placed into a syringe pump
(Cole-Parmer) modified with an aluminum base unit orienting the
pump vertically rather than horizontally (e.g., FIGS. 4-6). The
tube with the emulsion oil was aligned on the stir plate so that it
was centered below the plastic syringe needle and the magnetic stir
bar was spinning properly. The syringe pump was set to dispense 0.6
ml at 5.5 ml/hr. The PCR-bead solution was added to the emulsion
oil in a dropwise fashion. Care was taken to ensure that the
droplets did not contact the side of the tube as they fell into the
spinning oil.
Once the emulsion was formed, great care was taken to minimize
agitation of the emulsion during both the emulsification process
and the post-emulsification aliquotting steps. It was found that
vortexing, rapid pipetting, or excessive mixing could cause the
emulsion to break, destroying the discrete microreactors. In
forming the emulsion, the two solutions turned into a homogeneous
milky white mixture with the viscosity of mayonnaise. The contents
of the syringe were emptied into the spinning oil. Then, the
emulsion tube was removed from the holding jig, and gently flicked
with a forefinger until any residual oil layer at the top of the
emulsion disappeared. The tube was replaced in the holding jig, and
stirred with the magnetic stir bar for an additional minute. The
stir bar was removed from the emulsion by running a magnetic
retrieval tool along the outside of the tube, and the stir bar was
discarded.
Twenty microliters of the emulsion was taken from the middle of the
tube using a P100 pipettor and placed on a microscope slide. The
larger pipette tips were used to minimize shear forces. The
emulsion was inspected at 50.times. magnification to ensure that it
was comprised predominantly of single beads in 30 to 150 micron
diameter microreactors of PCR solution in oil (FIG. 7). After
visual examination, the emulsions were immediately amplified.
Example 13
Amplification
The emulsion was aliquotted into 7-8 separate PCR tubes. Each tube
included approximately 75 .mu.l of the emulsion. The tubes were
sealed and placed in a MJ thermocycler along with the 25 .mu.l
negative control described above. The following cycle times were
used: 1 cycle of incubation for 4 minutes at 94.degree. C.
(Hotstart Initiation), 30 cycles of incubation for 30 seconds at
94.degree. C., and 150 seconds at 68.degree. C. (Amplification),
and 40 cycles of incubation for 30 seconds at 94.degree. C., and
360 seconds at 68.degree. C. (Hybridization and Extension). After
completion of the PCR program, the tubes were removed and the
emulsions were broken immediately or the reactions were stored at
10.degree. C. for up to 16 hours prior to initiating the breaking
process.
Example 14
Breaking the Emulsion and Bead Recovery
Following amplification, the emulsifications were examined for
breakage (separation of the oil and water phases). Unbroken
emulsions were combined into a single 1.5 ml microcentrifuge tube,
while the occasional broken emulsion was discarded. As the emulsion
samples were quite viscous, significant amounts remained in each
PCR tube. The emulsion remaining in the tubes was recovered by
adding 75 of mineral oil into each KR tube and pipetting the
mixture. This mixture was added to the 1.5 ml tube containing the
bulk of the emulsified material. The 1.5 ml tube was then vortexed
for 30 seconds. After this, the tube was centrifuged for 20 minutes
in the benchtop microcentrifuge at 13.2K rpm (full speed).
After centrifugation, the emulsion separated into two phases with a
large white interface. The clear, upper oil phase was discarded,
while the cloudy interface material was left in the tube. In a
chemical fume hood, 1 ml hexanes was added to the lower phase and
interface layer. The mixture was vortexed for 1 minute and
centrifuged at full speed for 1 minute in a benchtop
microcentrifuge. The top, oil/hexane phase was removed and
discarded. After this, 1 ml of 80% Ethanol/1.times. Annealing
Buffer was added to the remaining aqueous phase, interface, and
beads. This mixture was vortexed for 1 minute or until the white
material from the interface was dissolved. The sample was then
centrifuged in a benchtop microcentrifuge for 1 minute at full
speed. The tube was rotated 180 degrees, and spun again for an
additional minute. The supernatant was then carefully removed
without disturbing the bead pellet.
The white bead pellet was washed twice with 1 ml Annealing Buffer
containing 0.1% Tween 20. The wash solution was discarded and the
beads were pelleted after each wash as described above. The pellet
was washed with 1 ml Picopure water. The beads were pelleted with
the centrifuge-rotate-centrifuge method used previously. The
aqueous phase was carefully removed. The beads were then washed
with 1 ml of 1 mM EDTA as before, except that the beads were
briefly vortexed at a medium setting for 2 seconds prior to
pelleting and supernatant removal.
Amplified DNA, immobilized on the capture beads, was treated to
obtain single stranded DNA. The second strand was removed by
incubation in a basic melt solution. One ml of Melt Solution (0.125
M NaOH, 0.2 M NaCl) was subsequently added to the beads. The pellet
was resuspended by vortexing at a medium setting for 2 seconds, and
the tube placed in a Thermolyne LabQuake tube roller for 3 minutes.
The beads were then pelleted as above, and the supernatant was
carefully removed and discarded. The residual Melt solution was
neutralized by the addition of 1 ml Annealing Buffer. After this,
the beads were vortexed at medium speed for 2 seconds. The beads
were pelleted, and the supernatant was removed as before. The
Annealing Buffer wash was repeated, except that only 800 .mu.l of
the Annealing Buffer was removed after centrifugation. The beads
and remaining Annealing Buffer were transferred to a 0.2 ml PCR
tube. The beads were used immediately or stored at 4.degree. C. for
up to 48 hours before continuing on to the enrichment process.
Example 15
Bead Enrichment
The bead mass included beads with amplified, immobilized DNA
strands, and empty or null beads. As mentioned previously, it was
calculated that 61% of the beads lacked template DNA during the
amplification process. Enrichment was used to selectively isolate
beads with template DNA, thereby maximizing sequencing efficiency.
The enrichment process is described in detail below.
The single stranded beads from Example 14 were pelleted with the
centrifuge-rotate-centrifuge method, and as much supernatant as
possible was removed without disturbing the beads. Fifteen
microliters of Annealing Buffer were added to the beads, followed
by 2 .mu.l of 100 .mu.M biotinylated, 40 base enrichment primer
(5'-Biotin-tetra-ethyleneglycol spacers
ccattccccagctcgtcttgccatctgttccctccagtctcag-3'; SEQ ID NO:5). The
primer was complimentary to the combined amplification and
sequencing sites (each 20 bases in length) on the 3' end of the
bead-immobilized template. The solution was mixed by vortexing at a
medium setting for 2 seconds, and the enrichment primers were
annealed to the immobilized DNA strands using a controlled
denaturation/annealing program in an MJ thermocycler. The program
consisted of the following cycle times and temperatures: incubation
for 30 seconds at 65.degree. C., decrease by 0.1.degree. C./sec to
58.degree. C., incubation for 90 seconds at 58.degree. C., and hold
at 10.degree. C.
While the primers were annealing, Dynal MyOne.TM. streptavidin
beads were resuspend by gentle swirling. Next, 20 .mu.l of the
MyOne.TM. beads were added to a 1.5 ml microcentrifuge tube
containing 1 ml of Enhancing fluid (2 M NaCl, 10 mM Tris-HCl, 1 mM
EDTA, pH 7.5). The MyOne bead mixture was vortexed for 5 seconds,
and the tube was placed in a Dynal MPC-S magnet. The paramagnetic
beads were pelleted against the side of the microcentrifuge tube.
The supernatant was carefully removed and discarded without
disturbing the MyOne.TM. beads. The tube was removed from the
magnet, and 100 .mu.l of enhancing fluid was added. The tube was
vortexed for 3 seconds to resuspend the beads, and stored on ice
until needed.
Upon completion of the annealing program, 100 .mu.l of annealing
buffer was added to the PCR tube containing the DNA capture beads
and enrichment primer. The tube vortexed for 5 seconds, and the
contents were transferred to a fresh 1.5 ml microcentrifuge tube.
The PCR tube in which the enrichment primer was annealed to the
capture beads was washed once with 200 .mu.l of annealing buffer,
and the wash solution was added to the 1.5 ml tube. The beads were
washed three times with 1 ml of annealing buffer, vortexed for 2
seconds, and pelleted as before. The supernatant was carefully
removed. After the third wash, the beads were washed twice with 1
ml of ice cold Enhancing fluid. The beads were vortexed, pelleted,
and the supernatant was removed as before. The beads were
resuspended in 150 .mu.l ice cold Enhancing fluid and the bead
solution was added to the washed MyOne.TM. beads.
The bead mixture was vortexed for 3 seconds and incubated at room
temperature for 3 minutes on a LabQuake tube roller. The
streptavidin-coated MyOne.TM. beads were bound to the biotinylated
enrichment primers annealed to immobilized templates on the DNA
capture beads. The beads were then centrifuged at 2,000 RPM for 3
minutes, after which the beads were vortexed with 2 second pulses
until resuspended. The resuspended beads were placed on ice for 5
minutes. Following this, 500 .mu.l of cold Enhancing fluid was
added to the beads and the tube was inserted into a Dynal MPC-S
magnet. The beads were left undisturbed for 60 seconds to allow
pelleting against the magnet. After this, the supernatant with
excess MyOne.TM. and null DNA capture beads was carefully removed
and discarded.
The tube was removed from the MPC-S magnet, and 1 ml of cold
enhancing fluid added to the beads. The beads were resuspended with
gentle finger flicking. It was important not to vortex the beads at
this time, as forceful mixing could break the link between the
MyOne.TM. and DNA capture beads. The beads were returned to the
magnet, and the supernatant removed. This wash was repeated three
additional times to ensure removal of all null capture beads. To
remove the annealed enrichment primers and MyOne.TM. beads, the DNA
capture beads were resuspended in 400 .mu.l of melting solution,
vortexed for 5 seconds, and pelleted with the Magnet. The
supernatant with the enriched beads was transferred to a separate
1.5 ml microcentrifuge tube. For maximum recovery of the enriched
beads, a second 400 .mu.l aliquot of melting solution was added to
the tube containing the MyOne.TM. beads. The beads were vortexed
and pelleted as before. The supernatant from the second wash was
removed and combined with the first bolus of enriched beads. The
tube of spent MyOne.TM. beads was discarded.
The microcentrifuge tube of enriched DNA capture beads was placed
on the Dynal MPC-S magnet to pellet any residual MyOne.TM. beads.
The enriched beads in the supernatant were transferred to a second
1.5 ml microcentrifuge tube and centrifuged. The supernatant was
removed, and the beads were washed 3 times with 1 ml of annealing
buffer to neutralize the residual melting solution. After the third
wash, 800 .mu.l of the supernatant was removed, and the remaining
beads and solution were transferred to a 0.2 ml PCR tube. The
enriched beads were centrifuged at 2,000 RPM for 3 minutes and the
supernatant decanted. Next, 20 .mu.l of annealing buffer and 3
.mu.l of two different 100 .mu.M sequencing primers
(5'-ccatctgttecctecctgtc-3'; SEQ ID NO:6; and
5'-cctatcocctgttgegtgtc-3' phosphate; SEQ ID NO:7) were added. The
tube was vortexed for 5 seconds, and placed in an MJ thermocycler
for the following 4-stage annealing program: incubation for 5
minutes at 65.degree. C., decrease by 0.1.degree. C./sec to
50.degree. C., incubation for 1 minute at 50.degree. C., decrease
by 0.1.degree. C./sec to 40.degree. C., hold at 40.degree. C. for 1
minute, decrease by 0.1.degree. C./sec to 15.degree. C., and hold
at 15.degree. C.
Upon completion of the annealing program, the beads were removed
from thermocycler and pelleted by centrifugation for 10 seconds.
The tube was rotated 180% and spun for an additional 10 seconds.
The supernatant was decanted and discarded, and 200 .mu.l of
annealing buffer was added to the tube. The beads were resuspended
with a 5 second vortex, and pelleted as before. The supernatant was
removed, and the beads resuspended in 100 .mu.l annealing buffer.
At this point, the beads were quantitated with a Multisizer 3
Coulter Counter (Beckman Coulter). Beads were stored at 4.degree.
C. and were stable for at least 1 week.
Throughout this specification, various patents, published patent
applications and scientific references are cited to describe the
state and content of the art. Those disclosures, in their
entireties, are hereby incorporated into the present specification
by reference.
Although particular embodiments have been disclosed herein in
detail, this has been done by way of example for purposes of
illustration only, and is not intended to be limiting with respect
to the scope of the appended claims, which follow. In particular,
it is contemplated by the inventors that various substitutions,
alterations, and modifications may be made to the invention without
departing from the spirit and scope of the invention as defined by
the claims.
SEQUENCE LISTINGS
1
7140DNAArtificial SequenceChemically Synthesized Primer Sequence
1gcttacctga ccgacctctg cctatcccct gttgcgtgtc 40240DNAArtificial
SequenceChemically Synthesized Primer Sequence 2ccattcccca
gctcgtcttg ccatctgttc cctccctgtc 40320DNAArtificial
SequenceChemically Synthesized Primer Sequence 3gcttacctga
ccgacctctg 20420DNAArtificial SequenceChemically Synthesized Primer
Sequence 4ccattcccca gctcgtcttg 20544DNAArtificial
SequenceChemically Synthesized Primer Sequence 5ccattcccca
gctcgtcttg ccatctgttc cctccctgtc tcag 44620DNAArtificial
SequenceChemically Synthesized Primer Sequence 6ccatctgttc
cctccctgtc 20720DNAArtificial SequenceChemically Synthesized Primer
Sequence 7cctatcccct gttgcgtgtc 20
* * * * *
References